Print Email Facebook Twitter Liquid chromatography/tandem mass spectrometry detection of covalent binding of acetaminophen to human serum albumin Title Liquid chromatography/tandem mass spectrometry detection of covalent binding of acetaminophen to human serum albumin Author Damsten, M.C. Commandeur, J.N.M. Fidder, A. Hulst, A.G. Touw, D. Noort, D. Vermeulen, N.P.E. TNO Defensie en Veiligheid Publication year 2007 Abstract Covalent binding of reactive electrophilic intermediates to proteins is considered to play an important role in the processes leading to adverse drug reactions and idiosyncratic drug reactions. Consequently, both for the discovery and the development of new drugs, there is a great interest in sensitive methodologies that enable the detection of covalent binding of drugs and drug candidates in vivo. In this work, we present a strategy for the generation and analysis of drug adducts to human serum albumin. Our methodology is based on the isolation of albumin from blood, its digestion to peptides by pronase E, and the sensitive detection of adducts to the characteristic cysteine-proline- phenylalanine (CPF) tripeptide by liquid chromatography/tandem mass spectrometry. We chose acetaminophen (APAP) as a model compound because this drug is known to induce covalent binding to proteins when bioactivated by cytochromes P450 to its reactive N-acetyl-p-benzoquinoneimine metabolite. First, by microsomal incubations of APAP in presence of CPF and/or intact albumin, in vitro reference adducts were generated to determine the mass spectrometric characteristics of the expected CPF adducts and to confirm their formation on pronase E digestion of the alkylated protein. When applying this methodology to albumin isolated from blood of patients exposed to APAP, we were indeed able to detect the corresponding CPF adducts. Therefore, this strategy could be seen as a potential biomonitoring tool to detect in vivo reactive intermediates of drugs and drug candidates, e.g., in the preclinical and clinical development phase. Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics. Subject Cytochrome P450ParacetamolSerum albuminArticleBiological monitoringCovalent bondDrug protein bindingHumanLiquid chromatographyMicrosomePriority journalTandem mass spectrometryAcetaminophenAcetylcysteineAnimalsBenzoquinonesChromatography, LiquidCysteineHumansIminesMicrosomes, LiverModels, BiologicalMolecular StructureOligopeptidesProtein BindingRatsReproducibility of ResultsSerum AlbuminTandem Mass SpectrometryChemicalsCAS: cytochrome P450, 9035-51-2Paracetamol, 103-90-2Serum albumin, 9048-46-8Acetaminophen, 103-90-2Acetylcysteine, 616-91-1BenzoquinonesCysteine, 52-90-4IminesN-acetyl-4-benzoquinoneimine, 50700-49-7Oligopeptides To reference this document use: http://resolver.tudelft.nl/uuid:d861620b-3441-415b-add5-d4b30d41e937 DOI https://doi.org/10.1124/dmd.106.014233 TNO identifier 240113 ISSN 0090-9556 Source Drug Metabolism and Disposition, 35 (8), 1408-1417 Document type article Files To receive the publication files, please send an e-mail request to TNO Library.